Biologically active complex

ABSTRACT

A biologically active complex comprising alpha-lactalbumin or a variant of alpha-lactalbumin which is in the apo folding state, or a fragment of either of any of these, and a cofactor which stabilizes the complex in a biologically active form, provided that any fragment of alpha-lactalbumin or a variant thereof comprises a region corresponding to the region of α-lactalbumin which forms the interface between the alpha and beta domains, and further provided that when the complex comprises native alpha-lactalbumin, the cofactor is other than C18:1:9 cis fatty acid. These complexes have therapeutic applications for example in the treatment of cancer and as antibacterial agents.

The present invention relates to biologically active complexes, inparticular complexes derived from alpha-lactalbumin α-lactalbumin), topharmaceutical compositions containing these as well as to their use intherapy, in particular as anti-cancer or antibacterial agents.

Biologically active complexes obtained from milk and particularly humanmilk, together with their use as antibacterial agents is described forexample in EP-0776214.

HAMLET (formerly known as MAL) is a molecular complex that induces invitro apoptosis selectively in tumour cells, but not in healthydifferentiated cells. The apoptotic activity of this variant fold wasdiscovered by serendipity, in a fraction of human milk casein obtainedby precipitation at low pH, and was purified by ion exchangechromatography, eluting as a single peak after 1M NaCl. The elute wasshown by spectroscopy to contain partially unfolded α-lactalbumin in anapo-like conformation (M. Svensson, et al, (1999) J Biol Chem, 274,6388-96), with native-like secondary structure, but lacking specifictertiary packing of the side chains. The link between apoptosisinduction and the folding change was proven by deliberate conversion ofnative α-lactalbumin to the apoptosis inducing form (M. Svensson, etal., (2000) Proc Natl Acad Sci USA, 97, 4221-6). HAMLET was shown tobind to the surface of tumour cells, to translocate into the cytoplasmand to accumulate in cell nuclei, where it causes DNA fragmentation (M.Svensson, et al., (2000) Proc Natl Acad Sci USA, 97, 4221-6).

It has also been found that other reagents and specifically lipid suchas oleic acid, are useful in the conversion of human α-lactalbumin toHAMLET (human α-lactalbumin made lethal to tumour cells). In particular,it has been reported previously that oleic acid (C18:1:9cis) is requiredfor HAMLET production (M. Svensson, et al., (2000) Proc Natl Acad SciUSA, 97, 4221-6).

Five of the seven oxygens that ligate the Ca²⁺ are contributed by sidechain carboxylates of Asp residues at positions 82, 87 and 88 and bycarbonyl oxygens of Lys 79 and Asp 84, and two water molecules supplythe remaining ligands (Acharya et al., 1991). The bound Ca²⁺ brings theα-helical region and the β-sheet in close proximity, and two disulfidebonds flanking the Ca²⁺ binding site, make this part of the moleculefairly inflexible.

α-Lactalbumin is the dominant protein in human milk, where it is presentat a concentration of 2 mg/ml (140 μM). The mature protein consists of123 amino acid residues (14.2 kDa). Its three dimensional structure hasbeen determined to 1.7 A resolution and it is a globular protein withfour α-helices (residues 1-34, 86-123) and a triple strandedanti-parallel β-sheet (residues 38-82), linked by four disulphide bonds(61-77; 73-91; 28-111 and 6-120) (K. R. Acharya, et al., (1991) J MolBiol, 221, 571-81). Binding of Ca²⁺ to a single very high affinity Ca²⁺binding site is required for the protein to maintain a nativeconformation. Five of the seven oxygens that ligate the Ca arecontributed by side chain carboxylates of Asp residues at positions 82,87 and 88 and by carbonyl oxygens of Lys 79 and Asp 84, and two watermolecules supply the remaining ligands. The bound Ca²⁺ brings theα-helical region and the β-sheet in close proximity, and two disulfidebonds flanking the Ca²⁺ binding site, make this part of the moleculefairly inflexible.

The protein adopts the so called apo state found in HAMLET when exposedto low pH, or in the presence of chelators, that release the stronglybound Ca²⁺ ion (D. A. Dolgikh, et al., (1981) FEBS Lett, 136, 311-5; K.Kuwajima, (1996) Faseb J, 10, 102-09).

The applicants have found that the conversion of α-lactalbumin to HAMLETwith apoptotic activity, requires both a conformational or foldingchange and the presence of a lipid cofactor and this may preferably beachieved using a variant of alpha-lactalbumin. The conformational orfolding change is conveniently effected by removal of calcium ions, orby using a variant without calcium ions. However, once the change hasbeen effected, the presence of calcium or a functional calcium bindingsite does not result in any loss of activity.

Furthermore, they have found that the optimal cofactors for theconversion of alpha-lactalbumin to HAMLET are C18:1 fatty acids with adouble bond in the cis conformation at position 9 or 11. Saturated C18fatty acid or unsaturated fatty acids in the trans conformation, orfatty acids with shorter carbon chains did not form HAMLET, suggestingthat highly specific inter-molecular interactions are required forlipids to act as folding partners in this system.

According to the present invention there is provided a biologicallyactive complex comprising alpha-lactalbumin or a variant ofalpha-lactalbumin which is in the apo folding state, or a fragment ofeither of any of these, and a cofactor which stabilises the complex in abiologically active form, provided that any fragment ofalpha-lactalbumin or a variant thereof comprises a region correspondingto the region of alpha-lactalbumin which forms the interface between thealpha and beta domains, and further provided that when the complexcomprises native alpha-lactalbumin, the cofactor is other than C18:1:9cis fatty acid.

In particular the cofactors are selected from a cis C18:1:9 or C18:1:11fatty acid or a different fatty acid with a similar configuration.

The complex is suitably prepared by forming the apo conformation of theprotein, using conventional or molecular biological methods, and inparticular by removing calcium ions from alpha-lactalbumin or variants,or by using variants from which calcium ions have been released, orwhich do not have a functional calcium binding site.

However, the applicants have found that, once formed, the presence of afunctional calcium binding site, and/or the presence of calcium, doesnot affect stability or the biological activity of the complex.Biologically active complexes have been found to retain affinity forcalcium, without loss of activity.

Therefore complex of the invention may further comprise calcium ions butthe elimination of calcium from the complex is not essential, butprovides a convenient means for the preparation of the complex.

Thus, in a particular embodiment, there is provided a biologicallyactive complex which is obtainable by combining

-   (i) a cis C18:1:9 or C18:1:11 fatty acid or a different fatty acid    with a similar configuration; and-   (ii) alpha-lactalbumin from which calcium ions have been removed, or    a variant of alpha-lactalbumin from which calcium ions have been    released or which does not have a functional calcium binding site.;    or a fragment of either of any of these, provided that any fragment    comprises a region corresponding to the region of α-lactalbumin    which forms the interface between the alpha and beta domains, and    further provided that when (ii) is alpha-lactalbumin, (i) is other    than C18:1:9 cis fatty acid.

In particular, the complex will comprise elements (i) and (ii).

The expression “biological active” of “biological activity” as usedherein means that the complex has similar biological activity to thatreported for HAMLET. In other words, it will be effective in producingapopotosis in cancer cells and/or have antibacterial properties.

As used herein the expression “variant” refers to polypeptides orproteins which are homologous to the basic protein, which is suitablyhuman or bovine α-lactalbumin, but which differ from the base sequencefrom which they are derived in that one or more amino acids within thesequence are substituted for other amino acids. Amino acid substitutionsmay be regarded as “conservative” where an amino acid is replaced with adifferent amino acid with broadly similar properties. Non-conservativesubstitutions are where amino acids are replaced with amino acids of adifferent type. Broadly speaking, fewer non-conservative substitutionswill be possible without altering the biological activity of thepolypeptide suitably variants will be at least 60% identical, preferablyat least 70%, even more preferably 80% or 85% and, especially preferredare 90%, 95% or 98% or more identity.

When comparing amino acid sequences for the purposes of determining thedegree of identity, programs such as BESTFIT and GAP (both fromWisconsin Genetics Computer Group (GCG) software package). BESTFIT, forexample, compares two sequences and produces an optimal alignment of themost similar segments. GAP enables sequences to be aligned along theirwhole length and finds the optimal alignment by inserting spaces ineither sequence as appropriate. Suitably, in the context of the presentinvention when discussing identity of sequences, the comparison is madeby alignment of the sequences along their whole length.

The term “fragment thereof” refers to any portion of the given aminoacid sequence which will form a complex with the similar activity tocomplexes including the complete α-lactalbumin amino acid sequence.Fragment may comprise more than one portion from within the full-lengthprotein, joined together. Portions will suitably comprise at least 5 andpreferably at least 10 consecutive amino acids from the basic sequence.

Suitable fragments will be deletion mutants suitably comprise at least20 amino acids, and more preferably at least 100 amino acids in length.They include small regions from the protein or combinations of these.

In a particularly preferred embodiment, the variant used in the methodof the invention is one in which the calcium binding site has beenmodified so that the affinity for calcium is reduced, or it is no longerfunctional. It has been found that in bovine α-lactalbumin, the calciumbinding site is coordinated by the residues K79, D82, D84, D87 and D88.Thus modification of this site, for example by removing one of more ofthe acidic residues, can reduce the affinity of the site for calcium, oreliminate the function completely and mutants of this type are apreferred aspect of the invention.

The Ca²⁺-binding site of bovine α-lactalbumin consists of a 3₁₀ helixand an α-helix with a short turn region separating the two helices(Acharya K. R., et al., (1991) J Mol Biol 221, 571-581). It is flankedby two disulfide bridges making this part of the molecule fairlyinflexible. Five of the seven oxygen groups that co-ordinate the Ca²⁺are contributed by the side chain carboxylates of Asp82, 87 and 88 orcarbonyl oxygen's of Lys79 and Asp84. Two water molecules supply theremaining two oxygen's (Acharya K. R., et al., (1991) J Mol Biol 221,571-581).

Site directed mutagenesis of the aspartic acid at position 87 to alanine(D87A) has previously been shown to inactivate the strongcalcium-binding site (Anderson P. J., et al., (1997) Biochemistry 36,11648-11654) (FIG. 1 a) and the mutant proteins adopted to theapo-conformation.

Therefore in a particular embodiment, the aspartic acid residue at aminoacid position 87 within the protein sequence is mutated to a non-acidicresidue, and in particular a non-polar or uncharged polar side chain.

Non-polar side chains include alanine, glycine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine.A particularly preferred example is alanine.

Uncharged polar side chains include asparagine, glutamine, serine,threonine or tyrosine.

In order to minimize the structural distortion in the mutant protein,D87 has also been replaced by an asparagine (N) (Permyakov S. E., etal., (2001) Proteins Eng 14, 785-789), which lacks the non-compensatednegative charge of a carboxylate group, but has the same side chainvolume and geometry (FIG. 7 a). The mutant protein (D87N) was shown tobind calcium with low affinity (K-_(ca)2×10⁵M⁻¹) (Permyakov S. E., etal., (2001) Proteins Eng 14, 785-789).

Such a mutant forms a further preferred embodiment of the invention.

Thus particularly preferred variants for use in the complexes of theinvention are D87A and D87N variants of α-lactalbumin, or fragmentswhich include this mutation.

The region which forms the interface between the alpha and beta domainsis, in human α-lactalbumin, defined by amino acids 34-38 and 82-86 inthe structure. Thus suitable fragments will include these regions, andpreferably the entire region from amino acid 34-86 of the nativeprotein.

This region of the molecule differs between the bovine and the humanproteins, in that one of the three basic amino acids (R70) is changed toS70 in bovine α-lactalbumin thus eliminating one co-ordinating sidechain. It may be preferable therefore, that where the bovineα-lactalbumin is used in the complex of the invention, an S70R mutant isused.

It appears that three molecular events are required to form HAMLET fromα-lactalbumin. First, the tightly bound Ca²⁺-ion is released. Theapo-protein is then allowed to bind the lipid cofactor, for example, onan ion exchange matrix. Third, the active complex is eluted at high saltand dialysed. The elutes were characterised after repeating thisprocedure with 14 closely related fatty acids as shown hereinafter onlythe C18:1:9cis and C18:1:11cis complexes were found to cause apoptosis,and they alone gave distinct novel signals by NMR, indicating that theyformed a novel molecular complex. Several other fatty acids were capableof retaining the protein on ion exchange matrices and to stabilize theprotein in a partially unfolded conformation, but they did not formbiologically active complexes and gave sharper NMR signals as expectedfrom a mixture of protein and fatty acid.

It appears that the unsaturated C18:1cis fatty acids have uniquestructural features, allowing them to form HAMLET fromapo-α-lactalbumin, and suggest that they differ from the other fattyacids in that they offer the correct stereo-specific match. The lack ofsignificant HAMLET formation with a number of closely related fattyacids suggests a highly selective and specific process. Consequently,any other fatty acids used in the complex of the invention should haveessentially similar stereospecificity to these unsaturated C18:1cisfatty acids.

The Ca²⁺ binding site is 100% conserved in α-lactalbumin from differentspecies (Acharya K. R., et al., (1991) J Mol Biol 221, 571-581),illustrating the importance of this function for the protein. It isco-ordinated by five different amino acids and two water molecules. Theside chain carboxylate of D87 together with D88 initially dock thecalcium ion into the cation-binding region, and form internal hydrogenbonds that stabilise the structure (Anderson P. J., et al., (1997)Biochemistry 36, 11648-11654). A loss of either D87 or D88 has beenshown to impair Ca2+ binding, and to render the molecule stable in thepartially unfolded state (Anderson P. J., et al., (1997) Biochemistry36, 11648-11654).

Further, as reported hereinafter, mutant proteins with two differentpoint mutations in the calcium-binding site of bovine c-lactalbumin wereused. Substitution of the aspartic acid at position 87 by an alanine(D87A) totally abolished calcium binding and disrupted the tertiarystructure. After substitution of the aspartic acid by asparagine, theprotein (D87N) still bound calcium but with lower affinity and showed aloss of tertiary structure, although not as pronounced as for the D87Amutant (Permyakov S. E., et al., (2001) Proteins Eng 14, 785-789). Themutant protein showed a minimal change in packing volume as both aminoacids have the same average volume of 125A³, and the carboxylate sidechain of asparagines allow the protein to co-ordinate calcium, but lessefficiently (Permyakov S. E., et al., (2001) Proteins Eng 14, 785-789).Both mutant proteins were stable in the apo-conformation at physiologictemperatures but despite this conformational change they werebiologically inactive in the apoptosis assay. The results demonstratethat a conformational change to the apo-conformation alone is notsufficient to induce apoptosis.

The structure of α-lactalbumin is known in the art, and the preciseamino acid numbering of the residues referred to herein can beidentified by reference to the structures shown for example in Andersonet al. supra. and Permyakov et al supra.

Native bovine α-lactalbumin and the Ca²⁺ mutants could be converted tothe HAMLET like complex named BAMLET, showing that the same fatty acidstabilised bovine α-lactalbumin in the BAMLET conformation. Theconversion yield was lower, however, suggesting that lipid binding tothe bovine protein was less efficient. The structural basis for thiseffect is not clear. Bovine and human α-lactalbumin show 76% amino acidsequence identity and have similar native conformations(Wijesinha-Bettoni R., et al., (2001) J Mol Biol 312, 261-273). Thelower conversion yield for BAMLET suggested that the sequencedifferences influenced the fit between the fatty acid and the bovineprotein. The divergent sequences are mainly located in the α-helicalregion (A-helix 57%, B-helix 50%, C-helix 23% and 3₁₀-helix 25%difference) but this region is unlikely to be involved in fatty acidbinding (see Example 3 below).

The applicants believe that the lipid binding site in humanα-lactalbumin may be located in the groove between the α-helical andβ-sheet domains, which becomes exposed in the apo-protein. This regionof the molecule differs between the bovine and the human proteins, inthat one of the three basic amino acids (R70) is changed to S70 inbovine α-lactalbumin, thus eliminating one potential coordinating sidechain.

The results illustrated herein demonstrate that the change in biologicfunction requires not just a conformational change of the protein, butalso the lipid cofactor. The dual requirements for a change in proteinconformation and a lipid cofactor may be important to achieve tissuespecificity. The active complex should only be formed in localenvironments that favour the altered protein fold, and where lipidcofactors are available. In the case of HAMLET, such conditions arepresent in the stomach of the breast-fed child. The low pH precipitatescasein with α-lactalbumin in the apo-conformation, and activates pHsensitive lipases that release oleic acid from the milk phospholipids.It is interesting to note that α-lactalbumin and oleic acidrespectively, are the most abundant proteins and fatty acid in humanmilk. The lipids thus appear to function as “post-secretion chaperones”,involved in the adaptation of proteins to shifting external environment.The need for both a folding change and a tissue specific lipid makessense in order to protect tissues from the occasional protein foldingvariant on the loose, and to target the site where the novel function isneeded.

As part of the present study, the applicants tested the calcium affinityof the complexes. The obtained calcium affinity of α-lactalbumin washigher than previously reported (about 2 mm as compared to 10 mM andup), but the value found (0.15 M NaCl) falls in the range reported byothers at 0.1 M NaCl (Table 1 hereinafter). The difference may beexplained by the lower ionic strength as we observed that the Ca²⁺affinity of α-lactalbumin decreased by a factor of 200 between low andphysiological ionic strength. This is expected from salt screening ofelectrostatic interactions, and similar decreases have been obtainedwith other negatively charged proteins.

The decrease in K_(a) of α-lactalbumin at physiological salt may also bedue to competition between Na⁺ and Ca²⁺ for the same site, as Na⁺ hasbeen reported to bind to and stabilize α-lactalbumin in vitro (K_(a)=100M⁻¹ at 20° C.,). The lack of near-UV CD signals for D87A in the presenceof NaCl was a striking contrast to wild-type α-lactalbumin, suggestingthat Na⁺ binds to the Ca²⁺ site of the wild-type protein, and that theD87 side-chain is important also for coordination of this monovalention.

Complexes of the invention, as well as HAMLET, retained a high affinityfor Ca²⁺ at both low and physiological salt concentrations, showing thatthey can bind Ca²⁺ without loss of activity. This may appear surprising,as partially unfolded conformations of α-lactalbumin usually areassociated with the Ca²⁺-free state. Two possible explanations may beoffered. In the first and most likely scenario, α-lactalbumin convertsto HAMLET by unfolding and binding of oleic acid with little disturbanceof the α-helical domain. The Ca²⁺-binding site may then retain a similaraffinity as in the absence of oleic acid. A second possibility is thatthe Ca²⁺ site is disrupted and that the observed Ca²⁺ binding isexplained by the generation of a new Ca²⁺ site in HAMLET. The head groupof oleic acid might potentially coordinate calcium together with aminoacid residues. In this case, the classical Ca²⁺-binding site inα-lactalbumin would not be needed for Ca²⁺-binding to HAMLET. We findthis a less likely explanation, as D87A-BAMLET has the oleic acid boundto the unfolded protein, and thus should form the new Ca²⁺ site, butdoes not bind Ca²⁺. It appears therefore that the Ca²⁺-binding site isnot involved in the conversion of α-lactalbumin to anapoptosis-associated conformation, and that the structural changesassociated with Ca²⁺ binding to HAMLET do not hinder the biologicalfunction.

In HAMLET, α-lactalbumin retains a partially unfolded conformation aswell as a high affinity Ca²⁺ binding site. This apparent paradox shedsnew light on the molecular characteristics of α-lactalbumin in thecomplex. The X-ray structure of the native like apo form shows that thealpha and beta regions are largely intact, while the cleft between themis widened (Chrysina et al., J. Biol. Chem, (2000) 275, 37021-9). Asdiscussed above, the applicants believe that the cofactor such as oleicacid binds in the interface between the alpha and the beta domains, andthat the bound cofactor acid locks this region of the molecule, whileallowing the α-domain to maintain a native-like conformation. This issupported by the finding illustrated hereinafter that complexes of thistype such as HAMLET binds Ca²⁺ while retaining activity against tumorcells. It would appear therefore that HAMLET is therefore in a differentmolecular state than either the low salt apo α-lactalbumin or thenative-like apo form in physiological salt.

Complexes of the invention are useful in a variety of therapeuticapplications, including anti-cancer and antibacterial treatments, inparticular for treatment of infections of the respiratory tract. Forthese purposes, the complex is suitably formulated as a pharmaceuticalcomposition and these form a further aspect of the invention.

The complex can be administered in the form of an oral mucosal dosageunit, an injectable composition, or a topical composition. In any casethe protein is normally administered together with the commonly knowncarriers, fillers and/or expedients, which are pharmaceuticallyacceptable.

In case the protein is administered in the form of a solution or creamfor topical use the solution contains an emulsifying agent for theprotein complex together with a diluent or cream base. Such formulationscan be applied directly to the tumour, or can be inhaled in the form ofa mist into the upper respiratory airways.

In oral use the protein is normally administered together with acarrier, which may be a solid, semi-solid or liquid diluent or acapsule. Usually the amount of active compound is between 0.1 to 99% byweight of the preparation, preferably between 0.5 to 20% by weight inpreparations for injection and between 2 and 50% by weight inpreparations for oral administration.

In pharmaceutical preparations containing complex in the form of dosageunits for oral administration the compound may be mixed with a solid,pulverulent carrier, as e.g. with lactose, saccharose, sorbitol,mannitol, starch, such as potato starch, corn starch, amylopectin,cellulose derivatives or gelatine, as well as with an antifrictionagent, such as magnesium stearate, calcium stearate, polyethylene glycolwaxes or the like, and be pressed into tablets. Multiple-unit-dosagegranules can be prepared as well. Tablets and granules of the abovecores can be coated with concentrated solutions of sugar, etc. The corescan also be coated with polymers which change the dissolution rate inthe gastrointestinal tract, such as anionic polymers having a pka ofabove 5.5. Such polymers are hydroxypropylmethyl cellulose phthalate,cellulose acetate phthalate, and polymers sold under the trade markEudragit S100 and L100.

In preparation of gelatine capsules these can be soft or hard. In theformer case the active compound is mixed with oil, and the latter casethe multiple-unit-dosage granules are filled therein.

Liquid preparations for oral administration can be present in the formof syrups or suspensions, e.g., solutions containing from about 0.2% byweight to about 20% by weight of the active compound disclosed, andglycerol and propylene glycol. If desired, such preparations can containcolouring agents, flavouring agents, saccharine, and carboxymethylcellulose as a thickening agent.

The daily dose of the active compound varies and is dependant on thetype of administrative route, but as a general rule it is 1 to 100mg/dose of active compound at personal administration, and 2 to 200mg/dose in topical administration. The number of applications per 24hours depend of the administration route, but may vary, e.g. in the caseof a topical application in the nose from 3 to 8 times per 24 hours,i.e., depending on the flow of phlegm produced by the body treated intherapeutic use.

The invention further provides a method for treating cancer whichcomprises administering to cancer cells a complex or a composition asdescribed above.

The invention further provides a method for treating bacterialinfections which comprises administering to a patient in need thereof, acomplex or a composition as described above.

In the description, the following abbreviations have been used.

-   GC/MS: Gas chromatography/Mass spectrometry,-   EDTA: ethylenediamintetra acetic acid, Tris    tris(hydroxymethyl)aminomethane, ANS: 8-Anilinonaphtalene-1-sulfonic    acid,-   CD: circular dichroism,-   UV: ultra violet,-   NaCl: sodium chloride,-   NMR: nuclear magnetic resonance, ppm: parts per million-   FITC: fluorescein isothiocyanate,-   TLC: thin layer chromatography,-   DEAE: diethylaminoethyl,-   HCl: hydrochloric acid,-   EGTA: ethylene-bis(oxyethyleneitriol)tetraacetic acid.-   FPLC; fast protein liquid chromatography;-   PBS, phosphate-buffered saline.

The invention will now be particularly described by way of example withreference to the accompanying diagrammatic drawings in which:

FIG. 1 shows simplified fatty acid structures and in particular linedrawings of the unsaturated fatty acids, which were investigated fortheir ability to produce a HAMLET like molecular complex.C16:1:9cis=Palmitoleic acid, C18:1:6cis=Petroselinic acid,C18:1:9cis=Oleic acid, C18:1:11cis=vaccine acid, C20:1:11cis=Eicosenicacid, C18:1:9trans=Elaidic acid, C18:1:11trans=Trans vaccenic acid,C20:4,5,8,11,15cis=Arachidonic acid, C18:3:6,9,12cis=Gamma linolenicacid, C18:3:9,12,15cis=Linolenic acid, C18:2:9,12cis=Linolenic acid.

FIG. 2 is a series of graphs showing the retention of apo-α-lactalbuminon ion exchange matrices conditioned with individual fatty acids.

FIG. 3 illustrates tumour cell apoptosis induced by the lipid-proteincomplexes.

FIG. 4 shows the results of CD spectroscopy to determine the tertiarystructure of the fatty acid-protein complexes.

FIG. 5 shows the results of probing of the fatty acid-protein complexesby ANS spectroscopy as an indicator of hydrophobicity.

FIG. 6 shows the results of NMR analysis of complexes.

FIG. 7 illustrates the characterisation of the D87A and D87N mutants ofα-lactalbumin, in which panel A shows the structure of thecalcium-binding site.

FIG. 8 illustrates biological tests carried out using mutated proteinsalone, and shows that they do not induce apoptosis.

FIG. 9 illustrates the conversion of bovine α-lactalbumin to BAMLET,where panel A shows elution peaks obtained during the preparation,panels B and C relate to the biological testing of BAMLET, panel D showsthe results of near UV CD spectroscopy, panel E shows the results ofintrinsic fluorescence spectrometry, and panel F shows the ANS spectraof HAMLET and BAMLET.

FIG. 10 illustrates the production and test results for D87A and D87N toD87A-and D87N-BAMLET, where panel A shows elution peaks obtained duringthe preparation, panels B and C relate to the biological testing ofthese complexes, panel D shows the results of near UV CD spectroscopy,panel E shows the results of intrinsic fluorescence spectrometry, andpanel F shows the ANS spectra.

FIG. 11 shows the results of Ca²⁺ titrations in the presence of quin 2.The absorbance is shown as a function of total Ca²⁺ concentration forquin 2 mixed with human α-lactalbumin, HAMLET, or D87A-BAMLET, and quin2 alone. The solid lines are the fitted curves. The absorbance isnormalized using the fitted values for completely Ca²⁺-free andCa²⁺-loaded system, respectively.

EXAMPLE 1

Structural variants of oleic acid, and other fatty acids differing inthe degree of saturation, carbon chain length and cis/trans conformation(FIG. 1) were compared for their ability to form HAMLET-like complexesfrom apo-α-lactalbumin.

Apo α-lactalbumin was applied to column matrices that had beenpre-conditioned with each indicated fatty acid indicated in FIG. 1 usingthe method described by M. Svensson, et al., (2000) Proc Natl Acad SciUSA, 97, 4221-6, and the eluate after 1M NaCl was collected. Columnsconditioned with C18:1:9cis were used as a positive control (a). Allunsaturated fatty acids in the cis conformation retainedapo-α-lactalbumin on the column, but with varying efficiency.Unsaturated fatty acids in the trans conformation (C18:1, 9trans, C16:1,9trans) or saturated fatty acids (C6:0 and C18:0) failed to retainapo-α-lactalbumin on the column.

Results of ion exchange chromatography on fatty acid conditionedmatrices are shown in FIG. 2. Unsaturated C18 fatty acids in the cisconformation formed complexes with apo-α-lactalbumin. The C18:1:9cisfatty acid converted >90% of the added apo-α-lactalbumin. TheC18:1:11cis fatty acid was somewhat less efficient with a yield of about70%, and other unsaturated C18 cis fatty acids (C18:1:6cis,C18:2:9,12cis, C18:3:9,12,15cis and gamma C18:3:6,9,12cis gaveconsiderably lower yields.

Trans conformers of C18:1 and saturated fatty acids were practicallyinactive, however. Only small amounts of protein eluted with high saltfrom columns conditioned with C18:1:9trans, C18:1:11trans, or thesaturated C18:0 fatty acid and they were inactive.

Unsaturated cis fatty acids with shorter (C16:1:9cis) or longer(C20:1:11cis and C20:4:5,8,11,15 cis) carbon chains formed complexesthat eluted after 1M NaCl, with yields comparable to C18:1:11cis, butlower than C18:1:9cis. The columns conditioned with the saturated fattyacids C6:0, C14:0 or C16:0 retained no apo-α-lactalbumin.

The results demonstrate that apo-α-lactalbumin interacts in astereo-specific manner with C18:1 fatty acids, and that C18 fatty acidsmust be unsaturated and with the double bond in the cis conformation.Unsaturated fatty acids in the trans conformation were inactive, as werethe saturated fatty acids. Furthermore the results confirmed thatα-lactalbumin in its Ca²⁺-bound form bound only to a very low degree tothe C18:1 fatty acid conditioned column. The binding site for theunsaturated cis fatty acid that defines HAMLET is available only in theapo-conformation.

EXAMPLE 2

Biological Activity

Apoptosis induction was tested using the L1210 leukaemia cell line.Apoptosis induction in L1210 leukaemia cells exposed to the differentprotein-lipid complexes including the HAMLET control (M. Svensson, etal, (1999) J Biol Chem, 274, 6388-96). HAMLET and the C18:1:11ciscomplex had killed 99-100% of the cells after six hours (FIG. 3 Table),but the other complexes had little or no effect on the cell viability.Both C18:1cis fatty acid protein complexes induced DNA fragmentation(b), but the C18:1trans fatty acid complexes were inactive. The C16 andC20 unsaturated fatty acid complexes caused an intermediate degree ofDNA fragmentation, but no loss of cell viability.

L1210 cells were exposed to lipid extracts derived from HAMLET or fromeach of the other complexes. No effect on L1210 cell viability (FIG. 3Table) or DNA fragmentation (c) was detected after six hours exposure tolipid concentrations corresponding to the amount present in 1.0 mg ofprotein, even though 0.3 mg of HAMLET was sufficient to kill the cellsby apoptosis. At very high lipid concentrations, the cells died ofnecrosis but at no time were there evidence of apoptosis in response tolipids.

Thus the complexes formed with C18:1:9cis and C18:1:11cis fatty acidsinduced apoptosis more efficiently than the other protein lipidcomplexes (Table in FIG. 3). Cell viability was reduced from 99% to 0%in six hours at a concentration of 0.3 mg/ml, and DNA fragmentation wasobserved. Interestingly, other C18:1:cis protein-fatty acid complexeshad killed <50% of the cells at this time (Table in FIG. 3). TheC18:1:trans fatty acid complexes were inactive in the cellular assay, aswere the C20 fatty acid complexes, and the C16 fatty acids complexesshowed very low effects on cell viability.

The C18:1:cis fatty acid complexes had induced DNA fragmentation aftersix hours, suggesting that the cells were dying by apoptosis (FIG. 3 a).We were surprised to find evidence of DNA fragmentation also in somecells exposed to the other fatty acid complexes, even though these cellsremained viable at six hours.

These results demonstrate that the lipids do not trigger apoptosis, andthat HAMLET is defined by both the protein and the lipid. They furtherdemonstrate that only C18:1:9cis and C18:1:11cis, fulfil the criteriafor a cofactor in the formation of HAMLET, even though some of the otherfatty acids appeared to interact with apo-α-lactalbumin on theion-exchange matrix.

EXAMPLE 3

Structural Correlates of the Biologic Activity

The ability to stabilise the protein in an apo-like conformation wasdetermined by CD and ANS spectroscopy, and the structural integrationwas examined by NMR spectroscopy.

Conformation Assessed by CD Spectroscopy

The complexes eluting after 1M NaCl were examined by near UV CDspectroscopy (M. Svensson, et al., (2000) Proc Natl Acad Sci USA, 97,4221-6), using native or apo-α-lactalbumin and HAMLET as controls. Thenative α-lactalbumin control showed the characteristics of a well foldedprotein, with a minimum at 270 nm arising from tyrosine residues and amaximum at 294 nm arising from tryptophan residues. Theapo-α-lactalbumin control had lost most of the characteristic signals,indicating less restrained tyrosines and tryptophans. TheC18:3:9,12,15cis, C18:3:6,9,12cis, C20:4:5,8,11,15cis and C18:1:6ciscomplexes resembled HAMLET with a loss of signal in the tyrosine andtryptophan regions, while remaining complexes were similar to theapo-α-lactalbumin control.

HAMLET was shown to resemble apo-α-lactalbumin, but seems to retain evenless of the tertiary structure (FIG. 4 a). The other eluted fattyacid-protein complexes showed two main spectral patterns. TheC18:1:6cis, C18:3:9,12,15cis, C18:3,6,9,12cis and C20:4:5,8,11,15cisfatty acid complexes resembled HAMLET, while the C18:1:9trans,C18:1:11:cis or trans, C18:2:9,12cis and C16:1:9cis or trans complexeswere identical to apo control (FIG. 4 b-h). Unconvertedapo-α-lactalbumin that eluted in the void was shown to revert to thenative state in the presence of Ca²⁺.

These results indicate that all fatty acids, which retainapo-α-lactalbumin on the column, stabilise the protein in a partiallyunfolded conformation.

Exposure of Hydrophobic Surfaces, as Probed by ANS Spectroscopy

Apo-α-lactalbumin is known to expose hydrophobic side chains, due to themobility of the β-sheet. The complexes eluting after 1M NaCl wereexamined by ANS spectroscopy (M. Svensson, et al., (2000) Proc Natl AcadSci USA, 97, 4221-6) using native or apo-α-lactalbumin and HAMLET ascontrols. Native α-lactalbumin did not bind ANS as shown by the flatcurve and the low signal at 490 nm, but apo-α-lactalbumin showedsignificant ANS binding with enhanced intensity and a maximum at 470 nmas expected from the increased hydrophobicity of this fold. All of theα-lactalbumin-fatty acid complexes except C20:1:11cis bound ANS. TheC18:3:9,12,15cis, gammaC18:3:6,9,12cis and C18:1:6cis complexesresembled HAMLET, while the other fatty acid complexes showed evenhigher ANS binding.

The apo control showed the expected ANS binding with enhanced intensityand a maximum at 470 nm, while the native protein failed to bind ANS asshown by the flat curve and the low signal at 490 nm. HAMLET bound ANSwith a blue shift of the curve, but the peak was lower than forapo-α-lactalbumin, (FIG. 5 a).

The C18:1:6cis, C18:3:9,12,15cis, and C18:3:6,9,12cis fatty acidcomplexes, bound ANS with similar spectral intensity as HAMLET. Someother complexes (C18:1,9trans, C18:1,11cis and trans, C18:2,9,12cis,C16:1,9 cis and trans, C20:4,5,8,11,15cis) showed more intense ANSfluorescence than the apo-α-lactalbumin control. Finally, theC20:1:11cis complex did not bind any ANS (FIG. 5 b-h).

To exclude the direct binding of ANS to the fatty acids in theprotein-lipid complexes, mixtures of ANS to the fatty acid weresubjected to spectroscopy. No ANS—fatty acid interaction was observed(data not shown).

These results demonstrated that the C18:1:9cis fatty acid complexretains the ability to interact with ANS, but the lower intensitysuggested that the fatty acid in HAMLET might modify ANS binding. Thenegative results for C20:1:11cis complex suggested that the longer fattyacid hindered the interaction of hydrophobic surfaces in the proteinwith ANS.

¹H-NMR Spectroscopy

¹H-NMR was used to resolve the structural basis for the difference inactivity between the C18:1cis and the inactive protein-fatty acidcomplexes (FIG. 6).

Native α-lactalbumin showed the characteristics of a folded andwell-ordered protein with narrow lines and significant shift dispersion,a large number of sharp signals in the aromatic region (around 7 ppm)and several out shifted methyl signals (between 0.7 and −0.6 ppm). Theapo protein displayed narrow lines and significant shift dispersion withsignificant variations relative to the native state in the chemicalshifts of a large number of resonances. The aromatic and methylatedregions are shown in the left and right panels, respectively (M.Svensson, et al., (2000) Proc Natl Acad Sci USA, 97, 4221-6). HAMLETshowed broad lines and lack of out shifted methyl signals suggestive ofa partially unfolded state, and significantly different from the nativeprotein. The spectrum obtained with the C18:1,11cis protein complex wasvirtually identical to HAMLET but the spectra of the trans fatty acidcomplexes showed more narrow lines and out shifted signals suggestingthat the conformation of the C18:1:9cis or C18:1:11cis complexes areunique and that although the trans fatty acids bind toapo-α-lactalbumin, they do not alter the conformation so that HAMLET isformed.

The spectrum of both the C18:1,9cis and C18:1,11cis complexes showedbroad lines and little shift dispersion. The lines in the aromaticregion were clustered and there were no out shifted methyl signals below0.7 ppm. The broad signals of the fatty acid suggested that they formedan integral part of the complex.

The trans isomer complexes (C18:1:9trans and C18:1:11trans) differedmarkedly from the C18:1:9cis or C18:1:11cis complexes. Signals frombound fatty acid were detected, but they were smaller than for the ciscomplexes. The protein lines were narrow, and out shifted both in themethyl and the aromatic regions. These data suggested that the transfatty acids bind to apo-α-lactalbumin, but do not alter the conformationso that HAMLET is formed.

These results suggest that specific molecular interactions stabiliseapo-α-lactalbumin in the HAMLET conformation, and only the unsaturatedC18:1:9cis or C18:1:11cis fatty acids have the stereo specificproperties required to achieve this conformational switch.

Apo-α-lactalbumin differs from other known lipid-binding proteins inthat it contains both α-helical and β-sheet domains. The intracellularlipid-binding protein family have an all β-barrel structure, forming acavity which binds in a range of fatty acids varying in chain length andsaturation (J. Thompson, et al., (1997) J Biol Chem, 272, 7140-7150).Typically, the carboxylate head group of the fatty acids interacts withtwo to four positively charged amino acids, usually arginines, and thecarbon chain is co-ordinated by six to ten hydrophobic amino acids. Thecrystal structure of human serum albumin has revealed six asymmetricallydistributed, fatty acid binding sites within the repeating α-helicaldomain structure of the protein (S. Curry, et al., (1998) Nature StrucBiol, 5, 827-835). Each hydrophobic pocket is capped at one end by basicor polar side chains, co-ordinating the fatty acid head group. While thebinding of fatty acids to human serum albumin causes conformationalchanges with rotations of the three domains of the protein, andadjustments of side chains to make way for incoming fatty acid (S.Curry, et al., (1998) Nature Struc Biol, 5, 827-835), the molecule doesnot unfold or change function. We may therefore conclude that the lipidcofactor function in the conversion of α-lactalbumin HAMLET differs bothstructurally and functionally from these previously known protein lipidinteractions.

Tentative fatty acid binding sites were identified based on thethree-dimensional structures of native apo-α-lactalbumin. The nativeα-lactalbumin molecule is a hydrophilic, acidic protein, exposing mainlycharged and polar amino acids. Two hydrophobic regions are located inthe interior of the globular structure.

One is formed by residues from the C and D helices and the β-sheetdomain in the interface between the two domains. The second is formed byresidues in the A, B and 30₁₀-helices of the α-domain (FIG. 9) (L. C.Wu, et al., (1998) J Mol Biol, 280, 175-82; M. Saito, (1999) Proteinengineering 12, 1097-1104). The crystal and NMR structures of bovine apoα-lactalbumin have revealed a significant structural change in the cleftbetween the two domains (E. Chrysina, et al., (2000) J Biol Chem, 275,37021-37029; R. Bettoni-Wijesinha, et al., (2001) J Mol Biol, 307,885-898) upon Ca²⁺ release. The expansion of the Ca²⁺ binding loop tiltsthe 3₁₀ helix towards the C helix, resulting in a disruption of thearomatic cluster in the interface between the two domains (Trp 60 and104, Phe 53 and Tyr 103) (E. Chrysina, et al., (2000) J Biol Chem, 275,37021-37029). The α-domain, in contrast, remains structured in both thenative and the apo-conformations, with near native side chain packing.It seems likely therefore that the C18:1 fatty acid binds in theinterface between the α and β domains, and thus stabilises amolten-globule like conformation.

The shape of the hydrophobic pocket suggested that it should favourinteractions with bent molecules (FIG. 1). This may indeed explain theinability of the C18:1 trans conformers to form HAMLET. While fattyacids in this cis conformation are u-shaped around the double bond, withboth carbon chains projecting in one direction, trans fatty acids arerod shaped around the double bond due to the carbon chains on oppositesides of the double bond. The saturated fatty acids are most flexiblewith no structural constraints due to the lack of double bonds. Theresults thus indicate that only the cis conformation allows fatty acidsa close stereo-specific fit, and that the additional critical feature ofthe fatty acid is the carbon chain length. In addition, the pocket iscapped by basic residues, which may co-ordinate the polar head groups ofthe fatty acids, thus orienting the lipid. This interaction is, howevernot sufficient for activation as the trans and saturated fatty acids,which possess the same charged head group failed to form the activecomplex. It is highly likely that the stereo specific fit involves bothhydrophobic interactions with the lipid tail and electrostaticinteractions of the negatively charged head group with basic sidechains. Based on the analogy with other fatty acid binding proteins, thefatty acid may bind to HAMLET by electrostatic interactions between itsnegatively charged head group and basic side-chains in the protein, aswell as by van der Waal's contacts and hydrophobic effects with the tailthat are optimized with the preferred stereo specific match(C18:1:9cis).

EXAMPLE 4

Analysis of Variants of α-lactalbumin

The apo-conformation of α-lactalbumin is unstable and the proteinreverts to the native state at neutral pH and at the Ca²⁺ concentrationspresent in the apoptosis assay. In the HAMLET complex, the proteinmaintains an apo-like conformation, however. As the lipid alone does nottrigger apoptosis, it might act simply by stabilising theapo-conformation. A conformational change of the protein might then besufficient to induce apoptosisr but the unstable nature of the apoconformation has precluded experiments testing the activity of theprotein per se.

This question was addressed by site directed mutagenesis of the Ca²⁺binding site. The bound Ca²⁺ ion is co-ordinated by a constellation ofseven oxygen groups that form a distorted pentagonal bipyramid, but amutation of the Aspartic acid residue at position 87 is sufficient tofully or partially inactivate the Ca²⁺ binding site, generating mutantproteins locked in the apo-conformation (Anderson P. J., et al., (1997)Biochemistry 36, 11648-11654; Veprintsev D. B., et al., (1999) Proteins37, 65-72; Permyakov S. E., et al., (2001) Proteins Eng 14, 785-789).This study examined if apoptosis can be triggered solely by aconformational change from the native to the apo-state. Furthermore, theimportance of the Ca²⁺ binding site for the conversion to HAMLET wasinvestigated.

Native human α-lactalbumin was purified from human milk by ammoniumsulphate precipitation and phenyl sepharose chromatography as described(Svensson M., et al., (2000) Proc Natl Acad Sci USA 97, 4221-4226). Apoα-lactalbumin was generated from 25 mg of native α-lactalbumin dissolvedat 1.8 mM in Tris¹ (10M Tris/HCl pH 8.5) by addition of 5 mM EDTA toremove bound Ca²⁺. The conformational change was confirmed by near UV CDand ANS spectroscopy. Bovine α-lactalbumin was purchased from Sigma, St.Louis, Mo., USA and used without further purification.

In addition, a mutated bovine protein (D87A and D87N) was expressed inE. coli, purified, folded and lyophilised as described (Anderson P. J.,et al., (1997) Biochemistry 36, 11648-11654; Permyakov S. E., et al.,(2001) Proteins Eng 14, 785-789).

A column (14 cm×1.6 cm) packed with DEAE-Trisacryl M (BioSepra, France)was attached to a Bio-Logic chromatography system (Bio-Rad Laboratories,Hercules, Calif.), and eluted with a NaCl gradient (buffer A: 10 mMTris/HCl pH 8.5; buffer B: buffer A containing 1 M NaCl). The matrix wasconditioned with oleic acid (Larodan biochemicals, Malmo, Sweden). Tenmilligrams was dissolved in 500 μl 99.5% ethanol by sonication (3minutes using a Branson 2200 bath sonicator, Branson, Danbury, USA).After addition of 10 ml of 10 mM Tris/HCl, pH 8.5, the lipid solutionwas applied to a newly packed DEAE-Trisacryl M matrix and dispersedthrough out the matrix using a NaCl gradient.

Ten mg of each of human and recombinant and native bovine α-lactalbuminwas dissolved in 10 ml of 10 mM Tris/HCl pH 8.5 and added to the column.The protein fraction eluting after high salt was desalted by dialysis(Spectra/Pore, Spectrum Medical Industries, Laguna Hills, Calif.,membrane cut off 3.5 kDa) against distilled water with at least fourchanges of water in 100-fold volume excess, and then lyophilised.

The products were then subjected to spectroscopic analysis. The proteinsor protein fractions were dialyzed against doubly distilled water andlyophilised. Stock solutions were prepared by dissolving the lyophilisedmaterial in 10 mM potassium phosphate buffer at pH 7.5, andconcentrations determined as the absorbance at 280 nm (A/l=C (mg/ml)where 1 mg/ml of α-lactalbumin is 70 μM). The spectra were recorded atthe appropriate dilution.

Circular Dichroism (CD) spectra were obtained on a JASCO J-720spectro-polarimeter with a JASCO PTC-343 Peltier type thermostated cellholder. Quartz cuvettes were used with 1 cm path length and spectra wererecorded at 25° C. between 240 and 320 nm. The wavelength step was 1 nm,the response time δ s and the scan rate was 10 nm per minute. Six scanswere recorded and averaged for each spectrum. Baseline spectra wererecorded with pure buffer in the cuvette and substracted from theprotein spectra.

The mean residue ellipticity q_(m) (mdeg×cm²×dmol⁻¹) was calculated fromthe recorded ellipticity, q, asq _(n) =q/(c·n·1)where c is the protein concentration in M, n the number of residues inthe protein (123 in this case), 1 the path length in nm and q is theellipticy in degrees.

Fluorescence spectra were recorded at 25° C. on a Perkin Elmer LS-50Bspectrometer using a quartz cuvette with 1 cm excitation path length.Intrinsic (tryptophan) fluorescence emission spectra were recordedbetween 305 and 530 nm (step 1 nm) with excitation at 295 nm. Theexcitation bandwidth was 3 nm and the emission was 5 nm. ANSfluorescence emission spectra were recorded at 25° C. on a Perkin ElmerLS-50B spectrometer using a quartz cuvette with 1 cm excitation pathlength, between 400 and 600 nm (step 1 nm) with excitation at 385 nm.Both the excitation and emission bandpass were set to 5 nm. ANS ammoniumsalt (Fluka, Buchs, Switzerland) was added stepwise and the spectra at1.5 molar equivalents are shown.

The results are illustrated in FIG. 7. Panel A shows ribbon diagrams ofthe calcium-binding loop with the co-ordinating side chains shown asdarkly shaded lines. In the wild-type protein calcium is co-ordinated byK79, D82,84,87 (arrow) and D88. If D87 is changed to A (arrow), theprotein looses its ability to bind calcium. If D87 is changed to N(arrow) the protein can still bind calcium but with low affinity.

The results of the investigation into the tertiary structure of the twomutants is shown in panel B. Spectra were recorded in sodium phosphatebuffer without EDTA. Native bovine α-lactalbumin had a minimum at 270 nmarising from tyrosine residues and a maximum at 294 nm arising fromtryptophan residues. Apo α-lactalbumin showed the characteristic loss ofsignal, indicating less restrained tyrosines and tryptophans. The D87Amutant showed an almost complete loss of ellipticity consistent with apartially unfolded conformation. The spectrum of the D87N mutant showeddecreased ellipticity in the tyrosine and tryptophan regions, althoughnot to the same extent as the D87A mutant.

The result of intrinsic fluorescence spectroscopy is shown in panel C.Native bovine α-lactalbumin showed an intensity maximum at 335 nm and ashoulder at 320 nm, indicative of tryptophan residues in a foldedhydrophobic core, but shifted to 350 in apo α-lactalbumin indicatingthat the tryptophans are more accessible to the solvent. Both the D87Aand the D87N mutant showed intensity maxima at 350 nm resemblingapo-α-lactalbumin.

Fluorescence spectra at 1.5 equivalents of ANS are shown in FIG. 7 panelD. Native bovine α-lactalbumin did not bind ANS but resembled ANS addedto pure buffer. Apo α-lactalbumin bound ANS with a maximum at 475 nm andsignificantly enhanced intensity. The D87N and D87A mutants bound ANSwith intensity maxima at 475 nm, strongly resembling the spectrum of theapo control.

Thus in summary, the near UV CD spectroscopy, a nearly complete loss ofellipticity was observed demonstrating loss of tertiary structure (FIG.7 b) and the intensity maximum in the intrinsic tryptophan spectrum wasshifted towards higher wavelengths (FIG. 7 c). The D87A mutant bound ANSas shown by the height of the curve and the shift of the intensitymaximum to shorter wavelengths (FIG. 7 d). No spectral changes wereobserved following the addition of excess calcium (1 mM) showing thatthe D87A mutant did not bind calcium under these conditions (Anderson P.J., et al., (1997) Biochemistry 36, 11648-11654).

The near UV CD spectrum was intermediate between the native and the D87Aspectra, but had a significantly reduced ellipticity (FIG. 7 b) ascompared to native α-lactalbunim, and the intrinsic fluorescencespectrum was red shifted compared to native protein suggesting exposedtryptophans and a loss of tertiary structure (FIG. 7 c). The D87N mutantbound ANS demonstrating exposed hydrophobic surfaces (FIG. 7 d).Addition of EDTA (1 mM) had only a marginal effect on the near UV CDellipticity (Permyakov S. E., et al., (2001) Proteins Eng 14, 785-789),showing that the D87N mutant is in a molten globule like state also inthe presence of calcium.

It appears that the D87N protein is in an apo-like conformation, butwith better defined tertiary structure than D87A.

These results conformed that mutations in the calcium-binding site lockthe protein in a molten globule like conformation, which is insensitiveto the calcium conditions.

EXAMPLE 5

Bioassays of Apoptosis

The L1210 (ATCC, CCL 219) cell line was cultured in suspension, asdescribed (Svensson M., et al., (1999) J Biol Chem 274, 6388-6396). Thecells were harvested by centrifugation (200 g for 10 min), re-suspendedin cell culture medium (RPMI 1640 supplemented with 10% fetal calfserum, non essential amino acids, sodium pyruvate and 50 μggentamicin/ml, Life Technologies, Gibco BRL, Paisly, United Kingdom) andseeded into 24 well plates (Falcon, Becton Dickinson, N.J., USA) at adensity of 2×10⁶/well. The different agonists were dissolved in cellculture medium, without fetal calf serum, and added to the cells (finalvolume 1 ml per well). Plates were incubated at 37° C. in 5% CO₂atmosphere and 100 μl of fetal calf serum was added to each well after30 minutes. Cell culture medium served as a control.

Cell viability was determined by Trypan blue exclusion after six hoursof incubation. For analysis, 30 μl of the cell suspension was mixed with30 μl of a 0.2% trypan blue solution and the number of stained cells(dead cells) per 100 cells was determined by interference contrastmicroscopy (Ortolux II, Leitz Wetzlar, Germany).

DNA Fragmentation

Oligonucleosome length DNA fragments were detected by agarose gelelectrophoresis. The cell suspension remaining after trypan blue (970μl, 2×10⁶/ml) was lysed in 5 mM Tris, 20 mM EDTA, 0.5% Triton X-100 pH8.0 at 4° C. for 1 hour and centrifuged at 13,000×g for 15 minutes. DNAwas ethanol precipitated over night in −20° C., treated with proteinaseK and RNAse, loaded on 1.8% agarose gels and electrophoresed withconstant voltage set at 50V over night. DNA fragments were visualisedwith ethidium bromide using a 305 nm UV-light source and photographedusing Polaroid type 55 positive-negative film.

Mutant Proteins

The ability of the mutant proteins to induce apoptosis was tested usingthe L1210 cell line. The proteins were suspended in cell culture mediumat 2 mg/ml and the cell viability was determined after six hours ofincubation as was the DNA fragmentation. The HAMLET control inducedapoptosis at 0.3 mg/ml but the mutant proteins had no effect (FIG. 8).

FIG. 8, Panel A Table 1 shows the viability of L1210 cells after 6hours' exposure to the mutant proteins. The mutants were unable to killthe cells even at a concentration of 1.0 mg/ml (c.f the results forBAMLET (see FIG. 9) where the viability reduced from 98% to 4%.

The mutant proteins did not induce DNA fragmentation, but BAMLETstimulated the formation of the characteristic DNA ladder as shown inpanel B.

These results demonstrate that the protein without associated oleic acidis not sufficient to induce apoptosis in tumour cells.

EXAMPLE 6

Preparation of BAMLET (Bovine α-Lactalbumin Made Lethal to Tumour Cells)

In view of the structural homology between the human and bovine proteinsit should be possible to convert bovine α-lactalbumin to HAMLET likemolecule, with apoptosis inducing properties. Hence, bovineα-lactalbumin was subjected to the conversion conditions previously usedfor human α-lactalbumin. Bovine α-lactalbumin was treated with EDTA toremove Ca²⁺, subjected to ion exchange chromatography on a C18:1conditioned column and eluted with a NaCl gradient. A large portion ofthe applied material eluted in the void but about 40% formed a sharppeak after 1M NaCl (arrow FIG. 9 a). The eluate after high salt wassaved for analysis. Human α-lactalbumin was converted with higherefficiency.

A large proportion of the applied material eluted in the void (about60%), but a small sharp peak eluted after 1M NaCl (FIG. 9 a).

The apoptosis-inducing activity of the high salt peak, named BAMLET, wasinvestigated using the L1210 mouse leukemia cell line as describedabove. Loss of cell viability and DNA fragmentation were used as endpoints. The L1210 cells died rapidly when exposed to HAMLET (0.3 mg/ml)and DNA fragmentation was induced. The L1210 cells were equallysensitive to BAMLET.

BAMLET reduced cell viability from 99% to 12% at 0.3 mg/ml, after sixhours incubation and induces DNA fragmentation (FIG. 9 b). There was noapparent difference in efficiency of apoptosis induction between HAMLETand the bovine equivalent (FIG. 9 b).

The tertiary structure of BAMLET was assessed using near UV CDspectroscopy. Native bovine α-lactalbumin shared the characteristicspectrum of a well-folded protein with tyrosine dip and tryptophan peak,and native bovine α-lactalbumin did not bind ANS. The bovine apo proteinhad a reduced signal in both the tyrosine and tryptophan regions,indicative of a partially folded protein with flexible side chains, andsignificant ANS binding with the maximum at 470 nm and enhancedintensity. The bovine complex strongly resembled both HAMLET and theapo-control (FIG. 9 c). The native and apo controls were as in FIG. 7.HAMLET showed decreased ellipticity in the tyrosine and tryptophanregions characteristics of a partially unfolded protein. BAMLET hadspectra similar to the apo control and to HAMLET, indicating flexiblearomates.

The intrinsic fluorescence spectrum native bovine α-lactalbumin had anintensity maximum at 320 nm, as expected from tyrptophan residues in thefolded protein. The apo-protein had an intensity-maximum at 345 nm and ashoulder at 360 nm, indicating that the tyrptophans are more exposed(FIG. 9 e). HAMLET showed an intrinsic fluorescence intensity maximum at345 nm and a shoulder at 360 nm indicating solvent exposed tryptophans.The spectrum of BAMLET was similar to that of HAMLET but without theshoulder. The results indicate that tryptophan residues are shieldedfrom solvent in the native protein, but are more solvent exposed in theapo control, HAMLET and BAMLET.

The ANS spectrum of HAMLET was blue-shifted with the intensity maximumat 475 nm and increased quantum yield, indicative of ANS binding. Thespectrum of BAMLET was virtually identical to that of HAMLET. Theresults indicate exposed hydrophobic surfaces in the apo control, HAMLETand BAMLET but not in the native protein. The bovine complex and HAMLEThad virtually identical spectra and bound ANS (FIG. 9 f) resembling theapo control.

It can be concluded that bovine apo-lactalbumin can be converted in thepresence of C18:1 to a molecular complex that induces apoptosis, andnamed this complex BAMLET (Bovine α-lactalbumin made lethal to tumourcells).

EXAMPLE 7

Conversion of the D87A and D87N Mutants to BAMLET

The D87A mutant described-above was applied to a C18:1 conditioned ionexchange column without EDTA and the column, and most of the appliedprotein eluted as a sharp peak after high salt (arrow) (FIG. 10 a). TheD87N mutant was first treated with EDTA to remove residual calcium andapplied to the column. A small portion of applied protein eluted in thevoid volume, but the majority eluted as a sharp peak after high salt(arrow). The eluted protein-lipid complexes amounting to >90% of appliedD87A and >95% of the D87N protein were named D87A- and D87N-BAMLET.

These complexes were testing in the apoptosis and DNA fragmentationassays described above. Table III (FIG. 10 b) shows the loss ofviability after 6 hours' exposure of L1210 cells to d87A- andD87N-BAMLET. At 0.5 mg/ml D87A- and D87N-BAMLET reduced the viabilityfrom 98% and 13% and 17% respectively. The D87A-and D87N-BAMLET inducedDNA fragmentation similar to the BAMLET control (FIG. 10 c). The LD₅₀iesof the mutant complexes (0.4 mg/ml) were slightly higher than for BAMLETand HAMLET (0.2 mg/ml).

Spectroscopic Characterisation of the D87A- and D87N-BAMLET

The conformations of D87A and D87N-BAMLET were compared to native andapo-bovine α-lactalbumin and to BAMLET. Near UV CD spectroscopy wascarried out on the complexes, with the native, apo and BAMLET controlsas in FIGS. 7 and 9. The D87A-BAMLET spectrum was very similar to theunconverted D87A protein with virtually no ellipiticy showing thatD87A-BAMLET is in the apo configuration. The spectrum of D87N-BAMLET wasvirtually identical to that of BAMLET with reduced ellipiticy in boththe tyrosine and tryptophan region (FIG. 10 d).

Intrinsic tryptophan fluorescence spectroscopy was conducted with thenative, apo-α-lactalbumin and BAMLET controls as in FIGS. 1 and 3. Theresults (FIG. 10 e) with D87A- and D87N-BAMLET showed intensity maximaat 345 nm with shoulder at 355 nm strongly resembling BAMLET and thehuman apo-α-lactalbumin control, suggesting that tryptophans areaccessible to solvent.

ANS spectroscopy was conducted, with the native, apo- and BAMLETcontrols as in FIGS. 7 and 9. Both D87A- and D87N-BAMLET bound ANS, withspectra resembling BAMLET and the apo-α-lactalbumin control.D87A-D87N-BAMLET bound ANS with the intensity maximum shifted to 470 nmand an increased quantum yield compared to the native control,indicating exposed hydrophobic surfaces in all proteins (FIG. 10 f).

These results demonstrated that D87A- and D87N-BAMLET maintain thepartially folded state with structural and functional propertiesresembling HAMLET and BAMLET. Calcium removal prior to oleic acidtreatment was not required for the D87A mutant because the protein ismost likely free from bound calcium and largely rests in the apo form.

As the complexes maintained their biologic activity, it appears that afunctional calcium-binding site is not required for the apoptoticfunction of this complex.

EXAMPLE 8

Calcium Binding to Alpha-Lactalbumin and the HAMLET Complex

The chromophoric chelator quin 2 was obtained from Fluka Chemie AG,Buchs, Switzerland. Other chemicals were of highest obtainablelaboratory quality. To produce Ca²⁺-free buffers, membrane tubing (M.w.cutoff: 3500, Spectrum Medical Industries Inc., LA, Ca, USA, boiled fourtimes in doubly distilled water before use) was filled with 10 ml Chelex100 (Biorad, Richmond, Calif., USA), sealed, and stored in the solutionsto absorb Ca²⁺.

Native human α-lactalbumin was purified from human milk by ammoniumsulphate precipitation and phenyl sepharose chromatography as described(Svensson et al., PNAS. 2000, 97, 4221-6). Bovine α-lactalbumin was bothpurchased from Sigma, St. Louis, Mo., USA and purified from bovine milkusing ammonium sulphate precipitation and phenyl speharosechromatography (Svensson et al., supra.). The mutated proteins (D87A andD87N) were expressed in E. coli, purified, folded and lyophilised asdescribed (Anderson et al., 1997), (Permyakov et al., Protein Eng.(2001, 14:785-9). The purity of the protein was assayed by SDS-PAGE andagarose gel electrophoresis, and by NMR spectroscopy.

Apo human or bovine α-lactalbumin for Ca²⁺-binding studies was generatedby dissolving α-lactalbumin in doubly distilled water containing a10-fold molar excess of EGTA at pH 8.0; The sample was applied to a G-25gel filtration column after an aliquot of saturated NaCl(calcium-depleted) and eluted by doubly distilled water. The sample waspassed through the saturated NaCl to reduce binding of EGTA to theprotein, and protein free from both Ca²⁺ and EGTA eluted in the water.The residual calcium content was below 0.1 equivalents as estimated fromthe titration in the presence of quin2, as described below.

Calcium- and EDTA-free HAMLET was generated as described before, withthe following adaptions. All buffers were stored with chelex(preparation described above) on a tipping board for a minimum of fivedays before use. Only plastic vials were used. The FPLC system (BioradBiologic, Richmond, Calif., USA), including the 20 ml ion exchangecolumn and all tubings, was washed with 2 volumes of 100 mM EDTA, pH8.5, and then rinsed with at least 10 volumes of millipore water. Thiswas followed by 2 volumes of buffer before the application of oleicacid. EGTA-free apo alpha-lactalbumin (20 mg) in 80 ml calcium-freebuffer was applied to the column in 4 consecutive runs (20 ml in eachrun). The collected fractions were pooled, dialysed, lyophilized andchecked for activity on tumor cells as described above. The calciumcontent was assayed using atomic absorption spectroscopy or titration inthe presence of quin 2 (described below). The product was alsocharacterized by agarose electrophoresis and ¹H-NMR.

To study the Ca²⁺ binding equilibrium, the proteins listed in table 1including the D87A BAMLET obtained as described in Example 7, andHAMLET, was titrated with calcium in the presence of a chromophoricchelator, quin 2, for which the absorbance at 263 nm decreases (approx.85%) upon Ca²⁺-binding. The method relies on competition for calciumbetween the protein and chelator, and can be used to quantitate highaffinity sites (ca. 10⁷-5·10⁹ M⁻¹ at low salt, and ca. 5 10⁵-1·10⁸ M⁻¹at 0.15 NaCl) when quin 2 is the chelator. The exact concentration ofthe chelator solution (in the range of 25-30 μM) was calculated from theabsorbance at 239.5 nm in the presence of excess calcium (usingε_(239.5)=4.2×10⁴ L mol⁻¹ cm⁻¹ for quin 2). Lyophilised protein wasdissolved in calcium free (<1 μM Ca²⁺) chelator solution at aconcentration of 25-30 μM. The absorbance at 263 nm (A₂₆₃) was recordedfor the protein/chelator solution using a UV/Vis 920 spectrophotometer(GBC Scientific Equipment Pty Ltd, Victoria, Australia). Calciumsolution (3 mM CaCl₂ in 2 mM Tris/HCl, pH 7.5, with or without 0.15 MNaCl) was added in portions of 4 μl. A₂₆₃ was recorded after eachcalcium addition.

The titration was continued until no absorbance change was seen for thelast five additions. A more concentrated Ca²⁺ stock (10 mM) was used atthe end of the titrations of the proteins in 0.15 M NaCl. The data wasanalysed by least squares fitting directly to the measured quantity,absorbance versus total calcium concentration, using the CaLigatorsoftware (Andre and Linse, 2002).

The Ca²⁺ affinity of α-lactalbumin has been extensively studied, mainlyfor the bovine protein. The reported values (Table 1) vary between2.5×10⁶ and 5.7×10⁸ M⁻¹, probably reflecting the experimental conditionsin terms of temperature, buffer, ionic strength, salt, and proteinpreparation.

The titration curve was very sensitive to the differences in affinitybetween chelator and protein, so this ratio can be obtained from thedata with high precision. Normalized data (absorbance versus total Ca²⁺concentration) at low salt are shown in FIG. 11, and the resultingCa²⁺-binding constants for the proteins at physiological (0.15 M NaCl)and low (no added) salt are listed in Table 1. We found a Ka of 1.8·10⁹M⁻¹ at low salt (K_(D)=0.56·10⁻⁹ M). The Ca²⁺ ion bound more weakly atphysiological salt concentrations (0.15 M NaCl): K_(a)=8.3·10⁶ M⁻¹(K_(D)=1.2·10⁻⁷ M).

HAMLET and BAMLET were shown to bind Ca²⁺ (FIG. 11). The calcium-bindingconstant for HAMLET was K_(a)=5.9·10⁹ M⁻¹ (K_(D)=1.7·10⁻⁹ M) at low saltand K_(a)=5.3.10⁶ M⁻¹ (K_(D)=1.9·10⁻⁷ M) at physiological saltconcentrations. Ca²⁺-titration data for D87A-BAMLET showed no differencecompared to the titration of quin 2 alone (FIG. 11), confirming thatD87A-BAMLET has lost the functional Ca²⁺ binding site.

It appears that HAMLET maintains a high calcium affinity in both a lowand physiological salt environment. Hence, the calcium affinity is only3 times lower for HAMLET than for α-lactalbumin when no salt is addedand 1.6 times lower at physiological salt concentration. TABLE 1 Calciumassociation constants (K_(a)) for α-lactalbumin or HAMLET at differentconditions. First five rows: this work, mean of two experiments. ProteinK_(a) (M⁻¹) Buffer Salt pH T (K) Method α-lac 1.8 · 10⁹   2 mM —chelator human Tris/HCl α-lac 8.3 · 10⁶   2 mM 150 chelator humanTris/HCl mM NaCl HAMLET 5.9 · 10⁸   2 mM — chelator Tris/HCl HAMLET 5.3· 10⁶   2 mM 150 chelator Tris/HCl mM NaCl D87A- n.d.*   2 mM — chelatorBAMLET Tris/HCl α-lac 4.3 · 10⁸  10 mM — 7.5 298 ITC bovine Tris/HClαlac 5.7 · 10⁸  10 mM — 7.5 298 ITC goat Tris/HCl α-lac 2.85 · 10⁸   10mM — 8 — DSC bovine Tris/HCl α-lac 4.8 · 10⁷  10 mM 7.8 EGTA, bovineAmmoinium Fluoroscence Bicarbonate α-lac 2.0 · 10⁷ H₂O 100 7 294 EDTA,⁴³Ca-NMR bovine mM KCl α-lac 2.0 · 10⁷ H₂O 100 7 294 EDTA, ⁴³Ca-NMRhuman mM KCl α-lac 2.7 · 10⁶  20 mM Tris — 7.5 298 1) Fluoescence bovine2) Hummel & Dryer method α-lac 1)   5 mM Tris, — 7.2 298 CD 270 nm 1)2.5 · 10⁸ 0.1 mM EDTA bovine 2) 2)   3 · 10⁸ human, 3) 3) goat 2.8 · 10⁸bovine 2.0 · 10⁷ H₂O 100 8 294- EGTA ¹³C-NMR mM 298 KCl bovine 1)  50 mMHepes — 8 1) fluorescence 2.0 · 10⁷ 310 2) 2) 4.0 · 10⁸ 293 bovine 2.5 ·10⁶  20 mM Tris — 7.5 298 microcalorimetry

1. A biologically active complex comprising alpha-lactalbumin or avariant of alpha-lactalbumin (α-lactalbumin) which is in the apo foldingstate, or a fragment of either of any of these, and a cofactor whichstabilises the complex in a biologically active form, provided that anyfragment of α-lactalbumin or a variant thereof comprises a regioncorresponding to the region of α-lactalbumin which forms the interfacebetween the alpha and beta domains, and further provided that when thecomplex comprises full length α-lactalbumin or a variant ofα-lactalbumin in which the calcium binding site has been modified sothat the affinity for calcium is reduced, or it is no longer functional,the cofactor is other than C18:1:9 cis fatty acid.
 2. A complexaccording to claim 1 wherein the cofactor is a cis C18:1:9 or C18:1:11fatty acid or a different fatty acid with a similar configuration.
 3. Acomplex according to claim 1 wherein the cofactor is C18:1:11 fattyacid.
 4. A complex according to claim 1 which comprises a fragment ofα-lactalbumin or a variant thereof, which fragment includes a regioncorresponding to the region of α-lactalbumin which forms the interfacebetween the alpha and beta domains.
 5. A biologically active complexaccording to claim 1 which is obtainable by combining (i) a cis C18:1:9or C18:1:11 fatty acid or a different fatty acid with a similarconfiguration; and (ii) α-lactalbumin from which calcium ions have beenremoved, or a variant of α-lactalbumin from which calcium ions have beenremoved or which does not have a functional calcium binding site; or afragment of either of any of these, provided that any fragment comprisesa region corresponding to the region of α-lactalbumin which forms theinterface between the alpha and beta domains, and further provided thatwhen (ii) is full length α-lactalbumin or a variant of α-lactalbumin inwhich the calcium binding site has been modified so that the affinityfor calcium is reduced, or it is no longer functional, (i) is other thanC18:1:9 cis fatty acid.
 6. A complex according to claim 1 which includesa variant of α-lactalbumin in which the calcium binding site has beenmodified so that the affinity for calcium is reduced, or it is no longerfunctional, and in which the cofactor is C18:1:11 fatty acid.
 7. Acomplex according to claim 6 wherein the variant has a mutation at aposition corresponding to at least one of the K79, D82, D84, D87 or D88residues.
 8. A complex according to claim 7 which includes a D87A orD87N variant of α-lactalbumin.
 9. A complex according to claim 1 whichcomprises a fragment of α-lactalbumin or a variant thereof, and wherethe fragment includes the entire region from amino acid 34-86 of thenative protein.
 10. A complex according to claim 1 wherein theα-lactalbumin is human or bovine α-lactalbumin or a variant of either ofthese.
 11. A complex according to claim 10 wherein the α-lactalbumin ishuman α-lactalbumin.
 12. A complex according to claim 11 wherein theα-lactalbumin is mutant bovine α-lactalbumin which includes an S70Rmutation.
 13. A complex according to claim 1 which further comprisescalcium ions.
 14. A pharmaceutical composition comprising a complexaccording to claim 1 in combination with a pharmaceutically acceptablecarrier.
 15. A method for treating cancer which comprises administeringto cancer cells a complex according to claim
 1. 16. (canceled)